1. Field of the Invention
This invention relates to a device for use in automating the detection of target nucleic acid sequences in biological-containing samples. The device described herein is for use with an automated process, including a fluid-delivery system and a thermal reaction chamber, as is described in U.S. patent application Ser. No. 07/227,348, filed Aug. 2, 1988 and now abandoned in favor of a continuing application Ser. No. 07/935,637, filed Aug. 24, 1992. The disclosure of the co-pending application is hereby incorporated herein by reference.
The invention in said co-pending application relates to a method and apparatus for automating the detection of target nucleic acid sequences in biological-containing samples involving a sequence of physical and chemical reactions, and more particularly to a system for the exposure of, amplification of, and labelled-probe coupling to, a specific, known nucleic acid sequence. The invention is especially suited to the automated detection of single, specific genetic sequences present at random in multiple samples containing biological material without labor-intensive DNA extraction and purification procedures being performed separately on each sample.
2. Description of the Related Art
Devices for receiving biological specimens for diagnostic purposes are varied and adapted to the methods of detection. The devices may take the form of tubes for liquid specimens, flat surfaces such as glass slides suitable for microscopy, microtiter dishes, Petri dishes and cubes containing growth medium, or filters made of various materials to which cell and viral components will adhere.
These specimen samples are then treated in such a way as to indicate either the presence or absence, or quantity, of a specific biological entity. Test reagents may either be preapplied to the device or added in series after the specimen is present. Test results are read manually by a technical person or automatically with instrumentation specifically designed for that assay. In some instances the specimen is diluted with a diluent, or an aliquot of the specimen is removed from the original collecting device and transferred to another vessel at some point in the assay. In some cases physical and chemical means are used to amplify the signal of the assay for greater sensitivity. Some assays require extraction or separation to isolate a specific component from other parts.
In DNA-based diagnostics the sequence specificity of base-pairing or enzymatic or other types of cleavage is exploited. The linear sequence of nucleotides in double-stranded DNA molecules forms the basis of replication of the genetic code. Hybridization is the binding of two single-stranded DNA strands whose base-pairing sequences are complementary. Temperature and salt concentration affect the stringency of these base-pairing matches. A change from high stringency to low stringency can cause the same DNA probe to be either exquisitely specific to detect a particular target or less specific and detect a group of related targets.
Wherever a unique, organism-specific polynucleotide sequence is identified, it is possible to use a labeled, synthetic molecule of the unique sequence to determine the presence of the organism by hybridization of the unknown sample to the labeled sequence. This detection method involves hybridization between DNA:RNA hybrids or DNA:DNA duplexes. The probe is a single-stranded nucleic acid molecule complementary to a unique nucleic acid sequence of the gene being tracked. The probe is labeled with an identifying molecule and introduced to the test sample. Hybridization has been an important research tool, but its use is limited to a few clinical laboratories because of the time, skill and knowledge required of the technician performing the test. DNA probes are being used as commercial diagnostics for a few infectious or genetic diseases, but their individual cost is prohibitive for mass screening.
While the common laboratory procedure for hybridization binds the target DNA to a solid support, an alternative approach is solution hybridization or hybridization which requires individual column separation of the unbound, labeled probe for each sample. This invention uses a gel matrix as a solid support. It is not necessary to transfer DNA to a membrane filter after purification and amplification. This approach eliminates any loss of DNA that occurs during transfer. (Purrello and Balazs, 1983, Anal. Biochem. 128:393-397).
Presently DNA preparation and amplification require labor-intensive protocols just as hybridization methods do. The only apparatus which automates DNA preparation is the Applied Biosystems Nucleic Acid Extractor, which will process sixteen tissue samples simultaneously in four hours. The sample must comprise homogenous tissue and already contain enough copies of the target DNA to be detected, i.e. about a million copies. The laboratory technician must then either fractionate the extracted DNA by gel electrophoresis or transfer the DNA to a solid support for detection by hybridization to a labeled probe. There is no laboratory apparatus or equipment currently on the market that automates hybridization so that it may be left unattended.
Suspending cells in agarose beads or cubes is a common laboratory procedure for preparing unsheared nucleic acid molecules for subsequent enzymatic modifications. (P. R. Cook, 1984, EMBO 3:1837-1842 and L. Van der Ploeg et al. 1984. Cell: 37:77-84). After solidification the agarose beads or cubes are subjected to extensive treatment with detergent, protease and salt. It is possible to remove all cellular constituents except DNA because the pores in the agarose matrix are large enough to allow rapid diffusion of proteins and other small macromolecules while quantitatively retaining genomic DNA (Smith and Cantor, 1986, Cold Spring Harbor Symposium on Qualitative Biology 51:115-122).
FMC Bioproducts, Rockland, Me., has a nonradioactive-label for DNA in which their product information states that the labeling is done directly in diluted, remelted agarose. This protocol allows electrophoretic fractionation of DNA in agarose and then quick and easy preparation of specific probes (Resolutions 1987 Newsletter 3(2):1-2). FMC also markets a new grade of agarose certified for reliable restriction endonuclease activity. Many other examples exist where research scientists are performing enzymatic modifications on DNA in agarose. D. Persons and O. Finn, (Biotechniques, 1986, 4:398-403) reported primer extension of cDNA on a poly A+ RNA template using a reverse transcriptase in remelted agarose. The method and device of this invention also involves primer extension with polymerase enzymes in agarose.
Immunodiagnostics are commonly used to identify organisms directly by antigenic determinants or to identify individuals by their antibodies which are produced by exposure to the antigen. The same problem is encountered with antigen identification as with DNA probes, i. e. the organism must be cultured if it is not present in sufficient numbers for detection. There is no in vitro method to amplify antibody- binding antigens accurately like there is with primer extension gene amplification. Low population targets in a mixed background cannot be identified immunologically. The gene amplification in vitro has given DNA probes the potential to outperform immunological detection. The accuracy, sensitivity and quantitation potential of DNA probes will make them the diagnostic of choice.
An automated system for simultaneously detecting target nucleic acid sequences from multiple samples must accommodate several different steps and varying reaction conditions. It must be constructed to change reagents and solvents automatically for each stage and monitor time, temperature and pH. If tests are automated and the same apparatus that performs one test for multiple samples in one run could be used for many different tests by changing a few selected reagents, the cost of gene detection would be relatively inexpensive and the system would supersede other methods because of its speed and preciseness.
In order to have enough gene copies for detection, present methods rely on selective cultivation of the organism which takes days to weeks depending upon the organism. A selective DNA amplification technique has been practiced whereby synthetic primers are annealed to single stranded or denatured, double-stranded nucleic acid target sequences and polymerase molecules incorporate nucleotides that replicate a portion of the nucleic acid extending from the primers. Using excess primers in pairs bordering a target sequence in a way that each polymerase extension includes sequences that are complementary to the other primer sequence is a method now termed polymerase chain reaction (PCR) (see U.S. Pat. Nos. 4,683,195 and 4,683,202). This method continues in repetitive rounds of replication until the target sequence has been amplified by a factor of more that 10 million. Saiki et al. reported that a thermostable DNA polymerase improves the specificity, yield, sensitivity and length of products that can be amplified (Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higuchi, G. T. Horn, K. B. Mullins, and H. A. Ehrlich, Science, 1988, 239:487-491). A selective gene amplification protocol that can duplicate a single copy of a nucleic acid target in vitro to a sufficient number of copies that can be detected over non-specific background binding with a labeled hybridization probe is the level of sensitivity that will enable easy screening of multiple samples. The accuracy of a gene detection is assured by labeling a probe complementary to a polynucleotide sequence between the two primer sequences for the purpose of hybridization identification. Thus, even if the primers had amplified non-target sequences because of duplicity of sequence or mismatch, the label would only be detected that bound to the target sequence.
The ability to amplify a single target DNA and/or RNA sequence enough to detect it without the cultivation of cells or organisms will simplify gene detection and attempts to automate it. Saiki et al. reported that PCR detects a single copy of target DNA present in one in 1.5 million cells. There is no reason to doubt that gene amplification by primer extension will detect a target DNA segment present at one copy per organism in the starting material. The ability to then quantify how many original copies or organisms there were per sample before amplification will make mass sampling and fate-monitoring possible by hybridization detection. Quantifying methods depend upon diluting the amplified gene so that individual signals are enumerated or intensity of total signal matches that of a known standard concentration.
Using the aforementioned gene amplification protocol, the presence of HIV-1 in peripheral blood mononuclear cells (PBMC) was determined by in situ hybridization to DNA from the PBMC's without prior cultivation of them (Ou, C., S. Kwok, S. Mitchell, D. Mack, J. Sninsky, J. Krebs, P. Feorino, D. Warfield, and G. Schochetman, Science, 1988, 239:295-297). This direct detection method reduces the time to three days from the three to four weeks required for cell cultivation and virus isolation. The polymerase chain extension technique started with DNA isolated from PBMC's, repetitively amplified the target DNA in solution, and analyzed bands on an autoradiogram produced by gel electrophoresis of restriction enzyme digests of the target DNA bound to end-labeled radioactive probe molecules.
In some instances the sizes of DNA fragments, produced by restriction endonuclease digestion or by amplification of a target sequences between primer pairs, are used to make a DNA-print for individual identification or aid in diagnosis of a genetic disease, cancer or infectious disease. For example, electrophoresis may be used to size-fractionate different-sized nucleic acids which have been specifically cleaved or whose native length puts them in a distinguishable size-length class. In the electrophoresis method, a current is applied to DNA loaded at the cathodal end of a gel matrix, which causes the DNA to migrate towards the anodal end of the matrix. The electrophoretic mobility of DNA is dependent on fragment size and is fairly independent of base composition or sequence. Resolution of one size class from another is better than 0.5% of fragment size (Sealy P. G. and E. M. Southern. 1982. Gel electrophoresis of DNA, p. 39-76. In D. Rickwood and B. D. Hames (EDS.), Gel Electrophoresis of Nucleic Acids. IRL Press, London). This reference and all other publications or patents cited herein are hereby incorporated by reference.
Electrophoresis methods thus require a vessel to hold the matrix material and the biological specimens to be subjected to electrophoresis. Such vessels may mold the gel matrix during its formation and may hold it during processing.
Diffusion of reagents is faster where the ratio of the matrix surface area to matrix volume is greatest as in thin, flat matrices. Likewise, electrophoresis of macromolecules requires less voltage and is faster in ultra-thin matrices or tiny (glass) capillaries. In these aqueous matrices, the vessel is necessary to prevent evaporation and to add strength in handling. Existing vessels that enclose matrices impede rapid diffusion of reagents and molecular probes. Once the existing vessels are taken apart in processing, they cannot be put back together to continue automated processing.
Accordingly, the invention aims to provide a system for automated gene identification of multiple samples, which prepares nucleic acids in the samples for testing, sufficiently amplifies target nucleic acid sequences and accurately detects their presence or absence in the samples.
Accordingly, the invention aims to provide a vessel for the individual specimens to be contained.
Yet another object of the invention is to mold matrix material which is to contain the specimen.
A further object of the invention is to carry the specimen in transport from the point of collection to the processing point.
A further object of the invention is to provide support of the specimen, embedding it in a matrix for automated processing.
A further object of the invention is to provide a convenient way to make the particles containing target nucleic acids of a specimen in a matrix available for optimal signal detection.
A further object of the invention is to allow for saturating specimens quickly with a series of solutions or for drying them automatically.
A further object of the invention is to concentrate specimen nucleic acids, or amplified products thereof, for detection of their presence.
A further object of the invention is to provide such a system wherein airflow and heating regulate temperature and humidity.
A further object of the invention is provide a barrier to evaporation of solutions during processing.
A further object of the invention is a mechanism to change its configuration during processing of the specimen to adapt to processing conditions.
A further objective of the invention is to provide support for reading the test results.
A still further object of the invention is to permanently store the nucleic acids present in the specimen for possible retesting and serve as a permanent record of the test, if an archival record is desired.
Other objects and advantages of the invention will be more fully apparent from the ensuing disclosure and appended claims.